It is known that N-glycosylation plays an important role in determining the biological activity and pharmacokinetic properties of many biopharmaceuticals. N-glycosylation of immunoglobulin G (IgG) at aspargine residue 297 plays a critical role in antibody stability and immune cell-mediated Fc effector function. For example, the presence of glycan as well as glycan structure itself at this conserved glycosylation site of the IgG Fc domain is crucial for promoting interaction between mAb and the Fc Receptor (FcR). It was shown that different forms of glycosylation (e.g. different glycan structures) define different effects, some are beneficial, while others are detrimental for biological and pharmacokinetic properties of mAbs. For example, defucosylated glycosylated therapeutic mAbs like Herceptin manufactured in CHO-cells (Shields, R. L., et al., 2002, J. Biol. Chem., 277: 26733-26740) or defucosylated anti-CD30 mAbs expressed in the small aquatic plant Lemna minor (Cox, K. M. et al., 2006, Nat. Biotechnol., 24:1591-1597) were shown to be ca 30-50-fold more active in the efficacy of Fc-gamma receptor IIIa (FcγRIIIa) mediated ADCC than their counterparts with alpha-1,6 (CHO cells) or alpha-1,3-linked (lemna) fucose residues. Similar results were reported for rituximab and other mAbs produced in animal and plant cells (Shinkawa, T. et al., 2003, J. Biol. Chem., 278: 3466-3473; Niwa, R., et al., 2004, Cancer Res., 64:2127-2133; Zeitlin, L., et al., 2011, Proc. Natl. Acad. Sci. USA, 108:20690-20694; Gasdaska, J R. et al., 2012, Mol. Immunol., 50:134-141).
Glycan absence at glycosylation sites of the Fc region has a detrimental effect on biological activities and pharmacokinetic properties of recombinant mAb. Removing the N-glycan severely impairs ADCC and CDC of CHO-cell produced mAbs (Jefferis R., 2007, Expert Opin Biol Ther., 7:1401-13). Aglycosylated cetuximab (an Epidermal Growth Factor Receptor inhibitor used for the treatment of metastatic colorectal cancer and head and neck cancer) did not bind to FcγRI or FcγRIIIa nor has it ADCC activity even at high effector-target cell ratios (Patel, D., et al., 2010, Hum Antibodies, 19:89-99). Structural studies suggested that the N-glycan might exert its effects predominantly through stabilisation of the conformation of the Fc domain (Mimura, Y. et al., 2000, Mol Immunol., 37:697-706; Sondermann, P., et al., 2000, Nature, 406:267-273; Mimura, Y. et al., 2001, J Biol Chem., 276:45539-45547). A recent detailed study demonstrated that hemi-glycosylation does not impact Fab-mediated antigen binding, nor does it impact neonatal Fc receptor binding. But hemi-glycosylated mAb-X has slightly decreased thermal stability in the CH2 domain and, more importantly, the hemi-glycosylated form shows significantly decreased binding affinity towards all Fc gamma receptors (FcγRs) including the high-affinity FcγRI, and the low-affinity FcγRIIA, FcγRIIB, FcγRIIIA and FcγRIIIB (Ha, S., et al., 2011, Glycobiology, 21:1087-1091). In the case mentioned above, the decreased binding affinities of hemi-glycosylated mAb-X to FcγRs result in a 3.5-fold decrease in antibody-dependent cell cytotoxicity (ADCC) in comparison to its fully glycosylated counterpart. As ADCC often plays an important role in therapeutic antibody efficacy, it is expected that glycosylation status will not only affect the antibody quality but also may impact the biological function of the product.
Commercially available therapeutic antibodies exhibit 99-100% Fc glycosylation site occupancy, while monoclonal antibody samples derived from transgenic plants may often contain significant amounts of aglycosylated variants (Karnoup, A. S., Kuppannan, K. & Young, S. A. 2007, J. Chromatogr. B, Analyt. Technol. Biomed. Life Sci., 859:178-191; Giorno, C., 2010, “Glycoengineering of monoclonal antibodies”, PhD Thesis, University Konstanz). Our internal data also revealed that plant-produced mAbs often contain up to 30% of aglycosylated N-glycosylation sites in the Fc domain. Such data in comparison with CHO-cell-produced mAbs indicate higher heterogeneity of the final plant-produced product and might have detrimental effect on biological activity and pharmaco-kinetik characteristics of plant-produced mAbs.
However, efficient interaction with Fc receptors of effector cells for triggering a patient's immune system via enhanced ADCC and CDC might not be crucial for some therapeutic mAbs including the ones for treating cancers. Effector cell activation might not be necessary for mAbs that are to be used as modulators (agonists or antagonists) of signal transduction. Also, because of glycosylation of Fc is necessary for maintaining monoclonal antibody conformation and stability (Zheng, K., Bantoq, C. & Bayer, R. 2011, Mabs, 3:568-576; Mimura, Y. et al., 2000, Mol Immunol., 37:697-706; Sondermann, P., et al., 2000, Nature, 406:267-273; Mimura, Y. et al., 2001, J Biol Chem., 276:45539-45547), complete removal of glycosilation sites e.g. by site-directed mutagenesis may not be an optimal solution.
The modulation of glycosylation site occupancy in the Fc region of IgG antibodies may be useful in cases where combination therapy is used with the help of antibody cocktails targeting immune activating and suppressing molecules (Kojima, T., et al., 2010, J. Immunol., 184:5493-5501), or, for example, in sequential treatment of HER2-positive cancers with tumor-targeting antibody trastuzumab, followed by treatment with a second antibody-agonist (anti-CD137) that activates the host innate immune system (Kohrt, H. E. et al., 2012, J. Clin. Invest., 122:1066-1075).
Therefore, it is an object of this invention to provide a process for modulating the occupancy of N-glycosylation sites in the Fc region of plant-expressed immunoglobulins and in other plant-expressed glycoproteins by glycans. It is also an object to provide a process of modulating the occupancy of N-glycosylation sites in the Fc region of plant-expressed immunoglobulins and in other plant-expressed glycoproteins to different levels, such as to more than 90% or almost 100% or to 50% or less by glycans. It is another object to provide recombinant glycoproteins such as immunoglobulins (notably IgGs) expressed in plants and having a desired glycan occupancy at N-glycosylation sites, notably at the N-glycosylation site in the Fc region of IgGs.